Small interfering RNA (siRNA) specifically degrades target complementary mRNAs in cells. The vector system that controls the expression of siRNAs has been widely used for academic research and clinical therapy. Although Pol III promoters are used to express small hairpin RNA to down-regulate genes in mammalian cells, the efficiency is not as potent as once hoped. It is also difficult to determine if cells express siRNA after transfection and it is hard to identify which cells express siRNAs. In this phase I study, we propose a novel Pol II promoter-controlled siRNA expression system. In this system, individual cells expressing siRNA can be traced and visualized by light microscopy. To detect expressing siRNA in cells more efficiently, we have also designed a new acrylamide-gel based isolation system that can enrich siRNAs (or any small RNA molecules) easily, quickly and with high efficiency. The new Pol ll-controlled siRNA expression vector and small RNA enrichment system has been devised and primarily tested. In this Phase I study, we will finally construct and optimize the siRNA expression vector and enrichment system. In phase II of the project, we will expand this new siRNA expression system to degrade specific mRNAs in a tissue- or cell type-specific manner. The specific aims of this Phase I feasibility study are: 1). Develop new Pol II promoter-controlled siRNA expressing vectors to down-regulate mRNA expressions 2). Develop a new small RNA isolation and enrichment system Successful completion of these goals will provide a novel system allowing researchers the ability to easily and verifiably down-regulate the expression of target genes. Furthermore, it will prove the feasibility of this approach and provide a firm foundation for the Phase II development of regulated knockdown strategies that will greatly enhance gene function studies. [unreadable] [unreadable]